There is a fundamental gap in understanding how the production of phenazines, known virulence factors, is transcriptionally regulated in the pathogenic bacterium, Pseudomonas aeruginosa. Continued existence of this gap represents an important problem because, until it is filled, understanding how and why phenazines are produced by P. aeruginosa and other pseudomonads will be largely incomprehensible. To address this, our long-term goal is to define the function(s) of PA2449 in the global physiology of P. aeruginosa. Specifically, it is imperative to know what genes and operons are regulated by PA2449, and what signals PA2449 responds to in order to activate transcription. The overall objective of this application will be to define the regulon of PA2449 in the medically relevant bacterial strain, P. aeruginosa, and determine its roles in the production of the phenazines virulence factors. The central hypothesis is that PA2449 encodes an enhancer binding protein (EBP) transcription factor that regulates the production of phenazines in P. aeruginosa. The hypothesis is based on our own preliminary findings, which were generated by comparing physiological and transcriptomic data for wild type P. aeruginosa PAO1 and a P. aeruginosa PA2449 transposon deletion strain. The rationale for the proposed research is that knowledge of the genetic network regulated by PA2449 will generate new strategies to prevent the expression of these virulence factors and thereby restrict the ability of P. aeruginosa to cause infection, particularly in the cystic fibrosis lung. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) Define the regulon of PA2449 and 2) Define the metabolic profile for PA2449. Under the first aim, techniques, which have been established as feasible in the applicant's hands, will be used; A) to perform microarray transcriptomic studies to define the regulon of PA2449; and B) to perform chromatin-immunoprecipitation-exonuclease (ChIP-exo) experiments to define the DNA binding sites of PA2449 throughout the genome of P. aeruginosa. Under the second aim, the investigators will use common HPLC and LC-MS-MS methodologies to assess the amino acid utilization of the PA2449 strain. The approach is innovative because it identifies and characterizes, for the first time, an EBP that specifically regulates the production of phenazines virulence factors in P. aeruginosa. The proposed research is significant, because it is expected to vertically advance and expand understanding of how and why phenazines are produced by P. aeruginosa. Ultimately, such knowledge has the potential to identify new targets to interfere with virulence factor production in P. aeruginosa.